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Cytomatrix 
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Engineering a Thymus for Efficient Production of Human T Cells |
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Deborah J. Marshall, Natalie Anosova, James Bagley, James Kinchsular, Vinh Hyunh, Michael Rosenzweig
Immunotherapy, Cytomatrix, Woburn, MA, 01801 David T. Scadden Experimental Hematology, Harvard-Massachusetts General Hospital, Boston, MA, 02114 |
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T lymphocytes are potentially valuable in the treatment of many diseases for which effective treatments or cures have not been found such as cancers; viral or bacterial infections amenable to therapy with cell vaccinations or therapeutics; diseases treatable by immune modulation, such as organ transplantation or autoimmunity; immune disorders and opportunistic infections arising from other treatments; drug-resistant and/or high frequency mutation pathogens; and inherited or acquired immunosuppression. The manufacture of T lymphocytes has proven problematic in part because of the obligate requirement that T cell generation is dependent on three-dimensional (3-D) interactions with thymic stromal cells. The lack of effective 3-D cell growth systems integrating these parameters has impeded the production of T cells for practical purposes. We have developed a novel artificial thymus culture system comprised of a stroma covered, three-dimensional, bio-compatible Cytomatrix™cell growth matrix. We have previously demonstrated that the Cytomatrix™ provided the obligate three dimensionality required for T cell differentiation; sustained the long-term growth of both thymic stromal cells and hematopoietic stem cells (HSC); and fostered the natural differentiation of T cells in the absence of exogenous factors (i.e. cytokines or serum). We are using this ex vivo culture system to generate T cells from hematopoietic progenitor cells isolated from adult human bone marrow in the context of human skin stroma to induce the differentiation of HSC to functional T cells for novel clinical uses. Briefly, human skin tissue was cultured in the Cytomatrix for 3 weeks. Lin- cells isolated from adult human bone marrow were then co-cultured on the stroma in the absence or presence of cytokines for an additional three weeks. FACS analysis demonstrated the development of CD4+CD8+ double positive as well as CD4+ and CD8+ single positive T cells, the majority of which were CD45RA+ (naive) and TCRab+. These T cells arose from de novo synthesis demonstrated by the presence of T cell receptor excisional circles (TREC) via PCR. Function was demonstrated by expansion in Con A and IL-2 supplemented media. These cells will be used in reconstitution assays to further test feasibility for clinical studies. |
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